1.Protective effect of asiaticoside on isoproterenol induced myocardial injury in mice
Ziyu Ma ; Xinmeng Zuo ; Zhenyu Wang ; Mingming Wang ; Cui Li
Acta Universitatis Medicinalis Anhui 2025;60(1):59-65
Abstract:
To study the protective effect of asiaticoside(AS) on Isoproterenol Hydrochloride(ISO)-induced myocardial injury in mice.
Methods:
Sixty male C57BL/6 mice were randomly divided into blank control(CON) group, model group [ISO,ISO 10/(kg·d)], Low dose group [ISO+AS-L,ISO 10 mg/(kg·d)+AS 5 mg/(kg·d)], Medium dose group [ISO+AS-M, ISO 10 mg/(kg·d)+AS 10 mg/(kg·d)], High dose group [ISO+AS-H, ISO 10 mg/(kg·d)+AS 20 mg/(kg·d)]. Heart mass ratio was counted; changes were observed in electrocardiogram; Enzyme linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-1β and cardiac troponin T(cTn-T) in serum; Masson staining was used to observe the fibrosis of mouse myocardial tissue; Western blot was used to detect the ratio of Bax and Bcl-2 protein expression levels(Bax/Bcl-2) and the expression levels of Caspase-3 and NLRP3 proteins in myocardial tissue; real-time quantitative polymerase chain reaction(qPCR) was used to detect the mRNA expression levels ofANP,BNP,β-MHC,TNF-α, IL-6, Type Ⅰ collagen(COLⅠ), and Type Ⅲ collagen(COLⅢ).
Results:
Compared with the CON group, the ISO group had an elevated heart-to-mass ratio(P<0.01), a lower heart rate(P<0.05), a prolonged QT interval(P<0.05), elevated expression of myocardial injury markers cTn-T,ANP,BNP, andβ-MHC(P<0.01); increased expression of IL-1β in the serum(P<0.01), increased expression ofTNF-αin the cardiac tissue and increasedIL-6expression(P<0.001), and NLRP3 protein expression was elevated(P<0.05); myocardium showed a large number of collagen fibers bluish staining(P<0.001),COLⅠ,COLⅢmRNA expression levels increased(P<0.001), and Bax/Bcl-2 ratio(P<0.001) and Caspase-3 expression were significantly elevated(P<0.05). Compared with ISO group, heart-to-mass ratio of mice in ISO+AS-L and ISO+AS-M groups decreased(P<0.05), heart rate increased, QT interval was shortened, cTn-T, ANP, BNP and β-MHC decreased(P<0.001), myocardial collagen fiber blue-staining decreased(P<0.01). The mRNA expression levels ofCOLⅠandCOLⅢdecreased(P<0.05). The expression levels of IL-1β and TNF-α decreased(P<0.01). NLRP3, Caspase-3 protein expression and Bax/Bcl-2 ratio decreased(P<0.05). The expression level ofIL-6in ISO+AS-M group decreased(P<0.01). The expression levels ofANP,BNP, andTNF-αmRNA expression were reduced in the ISO+AS-H group(P<0.001); the degree of myocardial fibrosis was improved(P<0.05), and the expression levels ofCOLⅠandCOLⅢmRNA were reduced(P<0.05).
Conclusion
AS has a protective effect against ISO-induced myocardial injury in mice by ameliorating cardiac fibrosis, inhibiting cardiomyocyte apoptosis and attenuating myocardial tissue inflammatory response.
2.Mechanism of Traditional Chinese Medicine Regulating JAK/STAT Signaling Pathway to Intervene in Lung Cancer: A Review
Jiarui CAO ; Bo FENG ; Chunzheng MA ; Weixia CHEN ; Jiangfan YU ; Shasha CAO ; Zhenyu ZHANG ; Wenhui OUYANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(9):265-276
Lung cancer is the malignant tumor with the highest incidence and mortality rates globally. Current treatment methods for lung cancer primarily include surgery, chemotherapy, targeted therapy, and immunotherapy. However, the main limitations of these treatments are their side effects, the drug resistance, and the economic burden they impose. As a critical cancer pathway, the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway regulates tumor occurrence and development through multiple mechanisms by influencing various downstream targets. Consequently, the JAK/STAT signaling pathway offers a promising avenue for lung cancer treatment research. Numerous studies have demonstrated that the JAK/STAT signaling pathway plays a key role in the proliferation and growth of lung cancer cells, angiogenesis, epithelial-mesenchymal transition (EMT), metabolic alterations, remodeling of the immune microenvironment, and the development of treatment resistance. Traditional Chinese medicine (TCM) has garnered increasing attention due to its minimal side effects, low economic burden, and its potential to enhance efficacy and reduce toxicity when used in conjunction with Western medicine. In addition to traditional Chinese medicine compounds, a growing number of Chinese medicine monomers have come into the spotlight because of their more targeted effects. Numerous studies investigating the regulation of the JAK/STAT signaling pathway by TCM in the treatment of lung cancer have demonstrated that TCM can inhibit the proliferation and invasion of lung cancer cells, tumor angiogenesis, and EMT, improve the inflammatory and immunosuppressive microenvironments, and enhance treatment sensitivity by intervening in the JAK/STAT signaling pathway, thereby impeding the progression of lung cancer. In recent years, the research on the regulation of this pathway by TCM in the treatment of lung cancer has been updated rapidly. However, the summary of these studies has not been updated in time. This review summarizes and reflects on the recent research findings regarding the regulation of the JAK/STAT signaling pathway by TCM to intervene in lung cancer from three aspects, introducing the JAK/STAT pathway, elaborating the mechanism of this pathway in lung cancer, and exploring the intervention of TCM in the treatment of lung cancer through this pathway, to provide more reference for the treatment of lung cancer in the future.
3.Laboratory malaria re-examination and evaluation of malaria diagnostic capability in Shanghai Municipal Malaria Diagnostic Reference Laboratory from 2017 to 2022
Yaoguang ZHANG ; Zhenyu WANG ; Min ZHU ; Li JIANG ; Qian ZHU ; Xiaojiang MA ; Qing YU ; Jian CHEN
Chinese Journal of Schistosomiasis Control 2024;36(5):521-526
Objective To evaluate the malaria diagnostic capability in Shanghai Municipal Malaria Diagnostic Reference Laboratory from 2017 to 2022 and to analyze factors affecting the diagnosis results, so as to provide the scientific evidence for increasing the laboratory malaria diagnostic capability during the post-elimination stage. Methods Plasmodium-negative blood smears were randomly sampled using a proportional sampling method each quarter during the period from 2017 to 2022 and scored by Shanghai Municipal Malaria Diagnostic Reference Laboratory. Malaria cases’ blood samples from district centers for disease control and prevention in Shanghai Municipality were re-reviewed using microscopy and multiplex PCR assay to evaluate the capability of malaria diagnosis. Results A total of 7 746 quality control blood smears were collected from district centers for disease control and prevention in Shanghai Municipality from 2017 to 2022, with a mean score of (76.74 ± 14.34) points and a qualification rate of 86.65% (6 712/7 746). A total of 387 blood smears were re-reviewed from 2017 to 2022, with an overall coincidence of 96.38% (373/387) for malaria diagnosis and 95.06% (308/324) for parasite species identification, and there were no significant differences in the coincidence for either malaria diagnosis (χ2 = 2.57, P > 0.05) or parasite species identification among years (χ2 = 1.04, P > 0.05). A total of 384 whole blood samples were collected from district centers for disease control and prevention, and the detection of whole blood samples was 70.31% (270/384) in district centers for disease control and prevention. All 384 whole blood samples were re-reviewed by Shanghai Municipal Malaria Diagnostic Reference Laboratory using a multiplex PCR assay from 2017 to 2022, with an overall coincidence of 94.07% (254/270) for malaria diagnosis and 99.55% (223/224) for parasite species identification, and there were no significant differences in the coincidence for either malaria diagnosis (χ2 = 5.77, P > 0.05) or parasite species identification among years (χ2 = 8.37, P > 0.05). The overall coincidence rates of Plasmodium-positive and negative whole blood samples were 100.00% (224/224) and 65.22% (30/46) in district centers for disease control and prevention, with a significant difference (χ2 = 82.82, P < 0.001), and there was a significant difference in the coincidence rate for identification of P. falciparum, P. vivax, P. malariae and P. ovale (χ2 = 24.28, P < 0.001). A total of 1 584 blind blood smears subjected to microscopic examinations by centers for disease control and prevention and medical institutions across all districts in Shanghai Municipality from 2017 to 2022, with a 96.15% (1 523/1 584) correct rate for malaria diagnosis and 85.07% (1 003/1 179) for parasite species identification, and there were significant differences in the correct rate of both malaria diagnosis (χ2 = 20.98, P < 0.001) and parasite species identification among years (χ2 = 70.77, P < 0.001). A total of 320 blind nucleic acid samples from malaria cases were tested, with a 99.38% (318/320) correct rate for malaria diagnosis and 100.00% (225/225) for parasite species identification, and there was no significant difference in the correct rate of malaria diagnosis among years (χ2 = 6.04, P > 0.05). Conclusions There were still shortcomings in blood smears preparation, microscopic examinations and nucleic acid testing in centers for disease control and prevention across all districts in Shanghai Municipality from 2017 to 2022. A greater role in the quality control of malaria diagnosis is recommended for Shanghai Municipal Malaria Diagnostic Reference Laboratory to prevent the re-establishment of imported malaria and consolidate the elimination achievements.
4.Study on the quality of Bolbostemmatis Rhizoma standard decoction by HPLC fingerprint combined with quantitative analysis of multi-components by single marker
Zhenyu LI ; Xiangyuan ZHOU ; Yifei MA ; Xian QIU ; Minyou HE ; Xiangdong CHEN ; Dongmei SUN
International Journal of Traditional Chinese Medicine 2024;46(1):76-83
Objective:To establish the HPLC fingerprint of Bolbostemmatis Rhizoma standard decoction; To determine the three effective components with similar structure by quantitative analysis of multi-components by single marker (QAMS); To evaluate the quality of Bolbostemmatis Rhizoma standard decoction.Methods:HPLC was adopted to establish the fingerprints of 15 batches of Bolbostemmatis Rhizoma standard decoction. The Chromatographic column was Waters XBridge Phenyl (4.6 mm×250 mm, 5 μm). The mobile phase was acetonitrile-0.1% phosphoric acid solution with gradient elution. Cluster analysis (HCA) and principal component analysis (PCA) were conducted based on the relative peak area of common peaks. The same method as the fingerprint was used to establish QAMS of tubeimoside A, B, C on Bolbostemmatis Rhizoma standard decoction.Results:There were 14 common peaks in the fingerprint of Bolbostemmatis Rhizoma standard decoction. It was confirmed that the peak 3 was L-tryptophan, the peak 11 was tubeimoside B, the peak 12 was tubeimoside C, and the peak 13 was tubeimoside A. 15 batches of Bolbostemmatis Rhizoma standard decoction from different origins were divided into 3 categories by HCA and PCA. There was no significant difference between QAMS and the external standard method (ESM) through the system suitability inspection. Conclusion:This method is accurate, reliable and has good specificity, which can effectively evaluate the quality of Bolbostemmatis Rhizoma standard decoction.
5.Factors influencing the severity of alcohol use disorder and the construction of risk prediction model
Xuezhi YANG ; Bing LU ; Wan WEI ; Zhen ZENG ; Sigui HU ; Yongkang CAO ; Zhenyu MA
Sichuan Mental Health 2024;37(2):131-136
BackgroundAlcohol use disorder (AUD) is a common chronic and relapsing psychiatric disorders. Identifying severe AUD early and intervening promptly is crucial to prevent irreversible harm. Currently, the assessment of AUD severity primarily relies on psychiatric examination by clinicians, and there is limited research on the factors influencing AUD severity and the development of prediction models. ObjectiveTo analyze the factors influencing AUD severity, and construct a risk prediction model to aid in the assessment of disease progression in AUD patients. MethodsA retrospective analysis was conducted on 1 358 first-time hospitalized patients admitted to Nanning Fifth People's Hospital from January 1, 2017 to December 31, 2022. These patients met the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) criteria for AUD. Basic patient data was collected, and the patients were divided into two groups based on disease severity: mild-moderate group (n=330) and severe group (n=1 028). The patients were randomly divided into training and test sets in a 7∶3 ratio. A Logistic regression model was constructed in the training set, and the predictive ability of the model for disease severity was evaluated using the receiver operating characteristic (ROC) curve in the test set. ResultsCompared with the mild-moderate group, the severe group had a higher proportion of patients living in urban areas (χ2=7.804), were farmers (χ2=17.991), had a higher frequency of alcohol consumption (more than 1 to 2 drinks/day) (χ2=35.267), had a higher age at first drinking (t=-3.858), had a greater number of comorbid somatic disorders (Z=-22.782), and had higher proportions of γ-Glutamyl transpeptidase (χ2=259.940) and total bilirubin abnormalities (χ2=148.552) (P<0.01). Logistic analysis conducted in the training set showed that being a farmer (OR=2.024, 95% CI: 1.352~3.029), having an older age at first drinking (OR=1.075, 95% CI: 1.025~1.129), drinking outside of mealtimes (OR=3.988, 95% CI: 2.408~6.606), having total bilirubin abnormalities (OR=1.034, 95% CI: 1.000~1.069), and having more comorbid somatic diseases (OR=4.386, 95% CI: 2.636~7.298) were identified as risk factors for disease severity in AUD patients. The area under curve (AUC) for this model in the test set was 0.906. ConclusionIn psychiatric hospitals, being a farmer, having an older age at first drinking, drinking outside of mealtimes, having abnormal total bilirubin levels, and having comorbidities with somatic illnesses may be risk factors for severe AUD.
6.Study on Anti-Depression Effect of Wenyang Jieyu Granules Through cAMP-PKA-CREB and Ras-MAPK Signaling Pathway in Rat Hippocampus
Shuang MENG ; Zhenyu FENG ; Xinxin WANG ; Xuyan WANG ; Xiaojuan MA ; Jianping ZHAO ; Jie ZHAO
World Science and Technology-Modernization of Traditional Chinese Medicine 2024;26(7):1802-1810
Objective To study the mechanism of Wenyang Jieyu Granules via cAMP-MAPK signaling pathway in hippocampus of chronic unpredictable mild stress(CUMS)model rats.Methods CUMS was used to establish depression model in rats.The depression state of rats was reflected by sucrose preference test,forced swimming test and open field test.The expressions of cAMP,phosphorylated extracellular signal-regulated kinase(p-ERK)and Raf protein kinase-1(Raf-1)in rat hippocampus were detected by enzyme-linked immunoassay(ELISA).Western blot was used to detect the expressions of cyclic adenosine phosphate response element binding protein(CREB),phosphorylated cyclic adenosine phosphate response element binding protein(p-CREB),extracellular signal-regulated kinase(ERK)and Mitogen-activatsion ofed Extracellular signal-regulated Kinas(MEK)in rat hippocampus.The mRNA expres cAMP,CREB,cyclic adenosine phosphate dependent protein kinase A(PKA),brain-derived neurotrophic factor(BDNF),and rat sarcoma(Ras)in rat hippocampus was detected by RT-PCR.The expression of caspase-3(caspase-3)in rath hippocampus was observed by immunohistochemistry.Results Compared with the model group,the body weight,sucrose preference rate and open field distance of model rats were increased in the positive control group and Wenyang Jieyu Granules group(P<0.05,P<0.01),and reduce the immobility time of forced swimming(P<0.05,P<0.01).It also increased the levels of cAMP,p-ERK,Raf-1,CREB,p-CREB,p-CREB/CREB,ERK and MEK in hippocampus of CUMS model rats(P<0.05,P<0.01).At the same time,the expression of cAMP,CREB,PKA,BDNF and Ras mRNA in the hippocampus of model rats were significantly improved(P<0.05,P<0.01),and he expression of caspase-3 in the hippocampus was reduced(P<0.05).Conclusion Wenyang Jieyu granules may exert antidepressant effect on model rats through cAMP and MAPK signaling pathways.
7.Interaction effect between vitamin D and gestational diabetes mellitus in the second trimester
Ziwen MA ; Xiaoying DING ; Liming CHU ; Yan ZHANG ; Aifeng ZHANG ; Yanjun ZHU ; Xun WANG ; Zhenyu XIE
Shanghai Journal of Preventive Medicine 2024;36(11):1094-1099
ObjectiveTo investigate the relationship between serum 25(OH)D and gestational diabetes mellitus in the second trimester (GDM), to analyze the interaction effect of key risk factors, so as to provide a basis for clinical personalized vitamin D supplementation. MethodsA total of 266 pregnant women who registered and took regular obstetric check-ups in Shanghai Pudong New Area Health Care Hospital for Women and Children from June to December 2022, were selected as the research subjects. According to the results of oral glucose tolerance test (OGTT), the subjects were divided into the GDM group (131) and control group (135). The level of serum 25(OH)D at the time of OGTT were detected and other clinical indicators were followed up. ResultsThe age, systolic blood pressure in early pregnancy, pre-pregnancy BMI, FPG, OGTT 1-hour and 2-hour glucose, GHb, HOMA-IR, TG, AST, Cr, D-D, FDP and SF at 35 weeks’ gestation of the pregnant women were higher in the GDM group than that of the pregnant women in the normal group, while gestational weight gain and serum 25(OH)D level were significantly lower than that of the pregnant women in the normal group. Serum 25(OH)D was negatively correlated with HOMA-IR and WBS’s, but positively correlated with TG and ALT. Serum 25(OH)D was non-linearly correlated with the risk of GDM in an inverted J-shape, and there was an interaction effect of advanced age, pre-pregnancy obesity and vitamin D deficiency on the risk of GDM. ConclusionVitamin D is non-linearly associated with the risk of GDM. The risk of GDM is significantly reduced when serum 25(OH)D level ≥30 ng∙mL-1.
8.Surveillance of parasitic infections in market-sold aquatic products and knowledge and practice towards food-borne parasitic diseases among residents in Shanghai Municipality from 2020 to 2023
Simin DAI ; Qing YU ; Zhenyu WANG ; Yaoguang ZHANG ; Jiawei YANG ; Zixin WEI ; Xiaojiang MA ; Qian ZHU ; Jian CHEN ; Hao PAN ; Huanyu WU
Chinese Journal of Schistosomiasis Control 2024;36(6):631-636
Objective To investigate the prevalence of parasitic infections in market-sold aquatic products in Shanghai Municipality, and to understand the knowledge and practice towards food-borne parasitic diseases among residents, so as to provide insights into the surveillance and control of food-borne parasitic diseases. Methods Freshwater products, seawater products and pickled products were randomly obtained from agricultural trade markets, supermarkets, retail stores and restaurants in Huangpu, Putuo, Minhang and Qingpu districts of Shanghai Municipality from 2020 to 2023. Parasite metacercariae and larvae were detected in these aquatic products using pressing method, digestion method and the dissection method, and the detection of parasitic infection was compared in different types of aquatic products. In addition, the knowledge and practice towards food-borne parasitic diseases were investigated among residents aged 10-80 years old who randomly selected from agricultural trade markets, supermarkets, community streets and other population concentration areas in these four districts by questionnaire in 2023, and the awareness of food-borne parasitic diseases knowledge and percentage of healthy behaviors formation were analyzed. Results A total of 2 109 aquatic products sampled from Huangpu, Putuo, Minhang and Qingpu districts of Shanghai Municipality from 2020 to 2023 were detected, and there were 317 products detected with parasitic infections, with an overall detection rate of 15.03%. There were 8 products detected with parasitic infections in 1 221 freshwater products, with a detection rate of 0.66%, and Clonorchis sinensis was the predominant parasite, which was detected in Pseudorasbora parva, Rhodeus amarus and Carassius auratus. There were 82 products detected with parasitic infections in 501 seawater products, with a detection rate of 16.37%, and Anisakis was the predominant parasite, which was detected in Trichiurus lepturus, Larimichthys polyactis, Pneumatophorus japonicus, Collchthys lucidus, Mugil cephalus, Larimichthys crocea, Scomberomorus niphonius, Stromateoides argenteus and Cololabis saira. There were 227 products detected with parasitic infections in 387 pickled products, with a detection rate of 58.66%, and the prevalence rates of Echinostoma metacercariae were 76.27% (225/295) in Bullacta exarata and 11.11% (2/18) in crab pastes, respectively (χ2 = 159.511, P < 0.05). No Paragonimus infection was found in freshwater shrimps, crabs and pickled products, and no Diphyllobothrium latum larvae infection was detected in freshwater and seawater fish. Questionnaire surveys showed that the awareness of food-borne parasitic diseases knowledge was 79.00% (222/281), and the percentages of washing hands before and after meals, not drinking filtered water or tap water directly, not eating raw or semi-raw food, being willing to change special dietary preferences or bad living habits for health, and being willing to learn more about food-borne parasitic diseases knowledge were 99.64% (280/281), 72.24% (203/281), 56.23% (158/281), 96.80% (272/281) and 97.51% (274/281) among residents living in Huangpu, Putuo, Minhang and Qingpu districts, respectively. Conclusions There are food-borne parasite contaminations in market-sold aquatic products in Shanghai Municipality. Although residents are aware of food-borne parasitic diseases knowledge, sustainable surveillance of food-borne parasitic diseases and improved health education are required to minimize the risk of human parasitic infections.
9.Relationship of physical activity and cognitive function to sleep quality in older adults with cognitive impairment: a mediating effect of resting EEG
Xiaochen MA ; Shufan LI ; Shuqi JIA ; Cong LIU ; Zhenyu ZHANG ; Dongyang HAN
Chinese Journal of Rehabilitation Theory and Practice 2024;30(12):1442-1451
ObjectiveTo investigate the relationship of physical activity (PA) and cognitive function to sleep quality in older adults with cognitive impairment based on resting electroencephalogram (EEG), and to explore the mediating role of specific EEG markers in the relationship between PA and sleep quality. MethodsFrom March to May, 2024, 137 older adults were recruited from Chenfu Jiayuan and Qiangwei Jiuli in Songjiang district, and Luyan communities in Jinshan district, Shanghai. The assessments included Montreal Cognitive Assessment (MoCA), International Physical Activity Questionnaire-Short Form (IPAQ-SF) and Pittsburgh Sleep Quality Index (PSQI), along with a five-minute EEG recording. ResultsThere was significant difference in sleep quality among older adults with different levels of cognitive impairment (t = -7.400, P < 0.001). The PSQI total score was negatively correlated with MoCA scores (r = -0.412, P < 0.001) and total physical activity level (PAL) (r = -0.363, P < 0.001). The θ power in the frontal areas (F3, F4) was significantly correlated with both PSQI scores and PAL (P < 0.01). The θ power in F3 + F4 exhibited a significant partial (effect size = -0.0004, 95%CI -0.0007 to -0.0002) mediating effect between PA and sleep quality in older adults with cognitive impairment. ConclusionOlder adults with more severe cognitive impairment tend to have poorer sleep quality, whereas higher PAL is associated with better sleep quality. PA can indirectly influence sleep quality in older adults with cognitive impairment by affecting θ power (F3 + F4).
10.Expression and correlation of CRP and IL-18,IL-37 in gingival crevicular fluid in the subjects treated with invisible correction without bracket and fixed aligners
Zhenyu ZHANG ; Yanning MA ; Bin ZHAO
Journal of Practical Stomatology 2024;40(6):823-828
Objective:To investigate the expression and correlation of C-reactive protein(CRP)with IL-18,IL-37 in gingival crevic-ular fluid(GCF)after invisible orthodontics treatment and fixed orthodontics treatment respectively.Methods:10 patients with invisible orthodontic treatment were included in the study group,and 12 patients with fixed orthodontic treatment were included in the control group.The expression levels of IL-18,IL-37 and CRP in GCF before,24 h,1 and 3 months after orthodontic treatment were detected by ELISA.Results:Before orthodontic treatment,there was no significant difference in the expression levels of IL-18,IL-37 and CRP in GCF between the 2 groups(P>0.05).The expression levels of IL-18,IL-37 and CRP in GCF increased at 24 h after treatment,and those in the study group were higher than in the control group(P<0.05).After 1 month of treatment,those in the study group were low-er than in the control group(P<0.05).After 3 months of treatment,the expression levels of the factors were not statistically different between the 2 groups(P>0.05).The leves of CRP,IL-18 and IL-37 in GCF were positivelly correlated in both groups(P<0.001).Conclusion:The levels of IL-18,IL-37 and CRP in GFC in the subjects treated with invisible correction are increased due to high stress stimulation in a period of 24 hours,suggesting that the effect on the periodontal tissue by the invisible correction may be greater than that by the fixed correction.


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